Conjugates useful in the treatment of prostate cancer

ABSTRACT

Chemical conjugates which comprise oligopeptides, having amino acid sequences that are selectively proteolytically cleaved by free prostate specific antigen (PSA), hydrophilic oligopeptide blocking groups and known cytotoxic agents are disclosed. Such conjugates are useful in the treatment of prostatic cancer and benign prostatic hypertrophy (BPH).

BACKGROUND OF THE INVENTION

In 1994 cancer of the prostate gland is expected to be diagnosed in200,000 men in the U.S. and 38,000 American males will die from thisdisease (Garnick, M. B. (1994). The Dilemmas of Prostate Cancer.Scientific American, April:72-81). Thus, prostate cancer is the mostfrequently diagnosed malignancy (other than that of the skin) in U.S.men and the second leading cause of cancer-related deaths (behind lungcancer) in that group.

Prostate specific Antigen (PSA) is a single chain 33 kDa glycoproteinthat is produced almost exclusively by the human prostate epithelium andoccurs at levels of 0.5 to 2.0 mg/ml in human seminal fluid (Nadji, M.,Taber, S. Z., Castro, A., et al. (1981) Cancer 48:1229; Papsidero, L.,Kuriyama, M., Wang, M., et al. (1981). JNCI 66:37; Qui, S. D., Young, C.Y. F., Bihartz, D. L., et al. (1990), J. Urol. 144:1550; Wang, M. C.,Valenzuela, L. A., Murphy, G. P., et al. (1979). Invest. Urol. 17:159).The single carbohydrate unit is attached at asparagine residue number 45and accounts for 2 to 3 kDa of the total molecular mass. PSA is aprotease with chymotrypsin-like specificity (Christensson, A., Laurell,C. B., Lilja, H. (1990). Eur. J. Biochem. 194:755-763). It has beenshown that PSA is mainly responsible for dissolution of the gelstructure formed at ejaculation by proteolysis of the major proteins inthe sperm entrapping gel, Semenogelin I and Semenogelin II, andfibronectin (Lilja, H. (1985). J. Clin. Invest. 76:1899; Lilja, H.,Oldbring, J., Rannevik, G., et al. (1987). J. Clin. Invest. 80:281;McGee, R. S., Herr, J. C. (1988). Biol. Reprod. 39:499). The PSAmediated proteolysis of the gel-forming proteins generates severalsoluble Semenogelin I and Semenogelin II fragments and solublefibronectin fragments with liquefaction of the ejaculate and release ofprogressively motile spermatoza (Lilja, H.. Laurell, C. B. (1984).Scand. J. Clin. Lab. Invest. 44:447; McGee, R. S., Herr, J. C. (1987).Biol. Reprod. 37:431). Furthermore, PSA may proteolytically degradeIGFBP-3 (insulin-like growth factor binding protein 3) allowing IGF tostimulate specifically the growth of PSA secreting cells (Cohen et al.,(1992) J. Clin. Endo. & Meta. 75:1046-1053).

PSA complexed to alpha 1-antichymotrypsin is the predominant molecularform of serum PSA and may account for up to 95% of the detected serumPSA (Christensson, A., Bjork, T., Nilsson, O., et al. (1993). J. Urol.150:100-105; Lilja, H., Christensson, A., Dahlén, U. (1991). Clin. Chem.37:1618-1625; Stenman, U. H., Leinoven, J., Alfthan, H., et al. (1991).Cancer Res. 51:222-226). The prostatic tissue (normal, benignhyperplastic or malignant tissue) is implicated to predominantly releasethe mature, enzymatically active form of PSA, as this form is requiredfor complex formation with alpha 1-antichymotrypsin (Mast, A. E.,Enghild. J. J., Pizzo, S. V., et al. (1991). Biochemistry 30:1723-1730;Perlmutter, D. H., Glover, G. I., Rivetna, M., et al. (1990). Proc.Natl. Acad. Sci. USA 87:3753-3757). Therefore, in the microenvironmentof prostatic PSA secreting cells the PSA is believed to be processed andsecreted in its mature enzymatically active form not complexed to anyinhibitory molecule. PSA also forms stable complexes with alpha2-macroglobulin, but as this results in encapsulation of PSA andcomplete loss of the PSA epitopes, the in vivo significance of thiscomplex formation is unclear. A free, noncomplexed form of PSAconstitutes a minor fraction of the serum PSA (Christensson, A., Björk,T., Nilsson, O., et al. (1993). J. Urol. 150:100-105; Lilja, H.,Christensson, A., Dahlén, U. (1991). Clin. Chem. 37:1618-1625). The sizeof this form of serum PSA is similar to that of PSA in seminal fluid(Lilja, H., Christensson, A., Dahlén, U. (1991). Clin. Chem.37:1618-1625) but it is yet unknown as to whether the free form of serumPSA may be a zymogen; an internally cleaved, inactive form of maturePSA; or PSA manifesting enzyme activity. However, it seems unlikely thatthe free form of serum PSA manifests enzyme activity, since there isconsiderable (100 to 1000 fold) molar excess of both unreacted alpha1-antichymotrypsin and alpha 2-macroglobulin in serum as compared withthe detected serum levels of the free 33 kDa form of PSA (Christensson,A., Björk, T., Nilsson, O., et al. (1993). J. Urol. 150:100-105; Lilja,H., Christensson, A., Dahlén, U. (1991). Clin. Chem. 37:1618-1625).

Serum measurements of PSA are useful for monitoring the treatment ofadenocarcinoma of the prostate (Duffy, M. S. ( 1989). Ann. Clin.Biochem. 26:379-387; Brawer, M. K. and Lange, P. H. (1989). Urol. Suppl.5:11-16; Hara, M. and Kimura, H. (1989). J. Lab. Clin. Med.113:541-548), although above normal serum concentrations of PSA havealso been reported in benign prostatic hyperplasia and subsequent tosurgical trauma of the prostate (Lilja, H., Christensson, A., Dahlén, U.(1991). Clin. Chem. 37:1618-1625). Prostate metastases are also known tosecrete immunologically reactive PSA since serum PSA is detectable athigh levels in prostatectomized patients showing widespread metatstaticprostate cancer (Ford, T. F., Butcher, D. N., Masters, R. W., et al.(1985). Brit. J. Urology 57:50-55). Therefore, a cytotoxic compound thatcould be activated by the proteolytic activity of PSA should be prostatecell specific as well as specific for PSA secreting prostate metastases.

It is the object of this invention to provide a novel anti-cancercomposition useful for the treatment of prostate cancer which comprisesoligopeptides having solubility augmenting oligopeptide blocking groupsin conjugation with a cytotoxic agent.

Another object of this invention is to provide a method of treatingprostate cancer which comprises administration of the novel anti-cancercomposition.

SUMMARY OF THE INVENTION

Chemical conjugates which comprise oligopeptides, having amino acidsequences that are selectively proteolytically cleaved by free prostatespecific antigen (PSA), hydrophilic oligopeptide blocking groups andknown cytotoxic agents are disclosed. Such conjugates are useful in thetreatment of prostatic cancer and benign prostatic hypertrophy (BPH).

DETAILED DESCRIPTION OF THE INVENTION

The instant invention relates to novel anti-cancer compositions usefulfor the treatment of prostate cancer. Such compositions comprise theoligopeptides covalently bonded directly, or through a chemical linker,to a cytotoxic agent. The oligopeptides are chosen from oligomers thatare selectively recognized by the free prostate specific antigen (PSA)and are capable of being proteolytically cleaved by the enzymaticactivity of the free prostate specific antigen. Such a combination of anoligopeptide and cytotoxic agent may be termed a conjugate.

The conjugates of the instant invention are further characterized byhaving a hydrophilic blocking group at the N-terminus of theoligopeptide which contributes to the aqueous solubility of theconjugate. Examples of such hydrophilic blocking groups include but arenot limited to hydroxylated and polyhydroxylated alkanoyl moieties andalkanoyl moieties that incorporate ether functionalities.

Ideally, the cytotoxic activity of the cytotoxic agent is greatlyreduced or absent when the oligopeptide containing the PSA proteolyticcleavage site is bonded directly, or through a chemical linker, to thecytotoxic agent and is intact. Also ideally, the cytotoxic activity ofthe cytotoxic agent increases significantly or returns to the activityof the unmodified cytotoxic agent upon proteolytic cleavage of theattached oligopeptide at the cleavage site.

Furthermore, it is preferred that the oligopeptide is selected fromoligopeptides that are not cleaved or are cleaved at a much slower ratein the presence of non-PSA proteolytic enzymes when compared to thecleavage of the oligopeptides in the presence of free enzymaticallyactive PSA.

For the reasons above, it is desirable for the oligopeptide to comprisea short peptide sequence, preferably less than ten amino acids. Mostpreferably the oligopeptide comprises seven or fewer amino acids.Because the conjugate preferably comprises a short amino acid sequence,the solubility of the conjugate may be influenced to a greater extent bythe generally hydrophobic character of the cytotoxic agent component.Therefore, the hydrophilic blocking groups of the instant conjugates areselected to offset or diminish such a hydrophobic contribution by thecytotoxic agent.

While it is not necessary for practicing this aspect of the invention, apreferred embodiment of this invention is a conjugate wherein theoligopeptide, and the chemical linker if present, are detached from thecytotoxic agent by the proteolytic activity of the free PSA and anyother native proteolytic enzymes present in the tissue proximity,thereby releasing unmodified cytotoxic agent into the physiologicalenvironment at the place of proteolytic cleavage. Pharmaceuticallyacceptable salts of the conjugates are also included.

It is understood that the oligopeptide that is conjugated to thecytotoxic agent, whether through a direct covalent bond or through achemical linker, does not need to be the oligopeptide that has thegreatest recognition by free PSA and is most readily proteolyticallycleaved by free PSA. Thus, the oligopeptide that is selected forincorporation in such an anti-cancer composition will be chosen both forits selective, proteolytic cleavage by free PSA and for the cytotoxicactivity of the cytotoxic agent-proteolytic residue conjugate (or, inwhat is felt to be an ideal situation, the unmodified cytotoxic agent)which results from such a cleavage. The term “selective” as used inconnection with the proteolytic PSA cleavage means a greater rate ofcleavage of an oligopeptide component of the instant invention by freePSA relative to cleavage of an oligopeptide which comprises a randomsequence of amino acids. Therefore, oligopeptide component of theinstant invention is a preferred substrate of free PSA. The term“selective” also indicates that the oligopeptide is proteolyticallycleaved by free PSA between two specific amino acids in theoligopeptide.

The oligopeptide components of the instant invention are selectivelyrecognized by the free prostate specific antigen (PSA) and are capableof being proteolytically cleaved by the enzymatic activity of the freeprostate specific antigen. Such oligopeptides comprise an oligomerselected from:

a) AsnLysIleSerTyrGln|Ser (SEQ.ID.NO.: 1),

b) LysIleSerTyrGln|Ser (SEQ.ID.NO.: 2),

c) AsnLysIleSerTyrTyr|Ser (SEQ.ID.NO.: 3),

d) AsnLysAlaSerTyrGln|Ser (SEQ.ID.NO.: 4),

e) SerTyrGln|SerSer (SEQ.ID.NO.: 5);

f) LysTyrGln|SerSer (SEQ.ID.NO.: 6);

g) hArgTyrGln|SerSer (SEQ.ID.NO.: 7);

h) hArgChaGln|SerSer (SEQ.ID.NO.: 8);

i) TyrGln|SerSer (SEQ.ID.NO.: 9);

j) TyrGln|SerLeu (SEQ.ID.NO.: 10);

k) TyrGln|SerNle (SEQ.ID.NO.: 11);

l) ChgGln|SerLeu (SEQ.ID.NO.: 12);

m) ChgGln|SerNle (SEQ.ID.NO.: 13);

wherein hArg is homoarginine, Cha is cyclohexylalanine and Chg iscyclohexylglycine.

In an embodiment of the instant invention, the oligopeptide comprises anoligomer that is selected from:

a) AsnLysIleSerTyrGln|SerSer (SEQ.ID.NO.: 14),

b) AsnLysIleSerTyrGln|SerAla (SEQ.ID.NO.: 15),

c) AlaAsnLysIleSerTyrTyr|Ser (SEQ.ID.NO.: 16),

d) AlaAsnLysAlaSerTyrGln|Ser (SEQ.ID.NO.: 17),

e) SerTyrGln|SerSerThr (SEQ.ID.NO.: 18),

f) SerTyrGln|SerSerSer (SEQ.ID.NO.: 19),

g) LysTyrGln|SerSerSer (SEQ.ID.NO.: 20),

h) hArgTyrGln|SerSerSer (SEQ.ID.NO.: 21),

i) SerTyrGln|SerSerLeu (SEQ.ID.NO.: 22);

j) SerTyrGln|SerLeu (SEQ.ID.NO.: 23);

k) SerChgGln|SerLeu (SEQ.ID.NO.: 24);

l) hArgChgGln|SerLeu (SEQ.ID.NO.: 25); and

m) hArgTyrGln|SerLeu (SEQ.ID.NO.: 26).

In a more preferred embodiment of the instant invention, theoligopeptide comprises an oligomer selected from:

GlyGluAsnGlyValGlnLysAspValSerGlnArgSerIleTyr|SerGlnThrGlu (SEQ.ID.NO.:27),

AlaSerTyrGln|SerSerLeu (SEQ.ID.NO.: 28);

SerhArgChgGln|SerLeu (SEQ.ID.NO.: 29);

hArgSerSerTyrGln|SerNle (SEQ.ID.NO.: 30);

hArgAlaSerChgGln|SerLeu (SEQ.ID.NO.: 31);

hArgSerSerTyrGln|SerLeu (SEQ.ID.NO.: 32);

hArgSerSerChg|SerLeu (SEQ.ID.NO.: 33);

SerhArgChgGln|SerLeu (SEQ.ID.NO.: 34);

hArgTyrGln|SerLeu (SEQ.ID.NO.: 35);

hArgSerSerChgGln|SerLeu (SEQ.ID.NO.: 36);

SerhArgTyrGln|SerLeu (SEQ.ID.NO.: 37);

SerSerTyrGln|SerLeu (SEQ.ID.NO.: 38);

SerSerSerChgGln|SerLeu (SEQ.ID.NO.: 39);

3PAL-SerSerChgGln|SerLeu (SEQ.ID.NO.: 40);

SerSerChgGln|SerLeu (SEQ.ID.NO.: 41);

SerSerSerChgGln|Ser(dLeu) (SEQ.ID.NO.: 42);

SerSerSerChgGln|SerVal (SEQ.ID.NO.: 43);

ProSerSerChgGln|SerVal (SEQ.ID.NO.: 44);

GlySerSerChgGln|SerLeu (SEQ.ID.NO.: 45);

hSerSerSerChgGln|SerLeu (SEQ.ID.NO.: 46);

hArgSerSerChgGln|SerNle (SEQ.ID.NO.: 47);

hArgTyrGln|SerSerSerLeu (SEQ.ID.NO.: 55);

LysTyrGln|SerSerSerLeu (SEQ.ID.NO.: 56);

SerTyrGln|SerSerSerLeu (SEQ.ID.NO.: 57);

SerSerChgGln-Ser(dLeu) (SEQ.ID.NO.: 58); and

3PAL-SerSerChgGln-Ser(dLeu) (SEQ.ID.NO.: 59); and

AlaSerChgGln-SerLeu (SEQ.ID.NO.: 60).

The phrase “oligomers that comprise an amino acid sequence” as usedhereinabove, and elsewhere in the Detailed Description of the Invention,describes oligomers of from about 3 to about 100 amino acids residueswhich include in their amino acid sequence the specific amino acidsequence described and which are therefore proteolytically cleavedwithin the amino acid sequence described by free PSA. Preferably, theoligomer is from 5 to 10 amino acid residues. Thus, for example, thefollowing oligomer: hArgSerAlaChgGln|SerLeu (SEQ.ID.NO.: 48); comprisesthe amino acid sequence: ChgGln|SerLeu (SEQ.ID.NO.: 12); and wouldtherefore come within the instant invention. It is understood that sucholigomers do not include semenogelin I and semenogelin II.

A person of ordinary skill in the peptide chemistry art would readilyappreciate that certain amino acids in a biologically activeoligopeptide may be replaced by other homologous, isosteric and/orisoelectronic amino acids wherein the biological activity of theoriginal oligopeptide has been conserved in the modified oligopeptide.Certain unnatural and modified natural amino acids may also be utilizedto replace the corresponding natural amino acid in the oligopeptides ofthe instant invention. Thus, for example, tyrosine may be replaced by3-iodotyrosine, 2-methyltyrosine, 3-fluorotyrosine, 3-methyltyrosine andthe like. Further for example, lysine may be replaced withN′-(2-imidazolyl)lysine and the like. The following list of amino acidreplacements is meant to be illustrative and is not limiting:

Original Amino Acid Replacement Amino Acid(s) Ala Gly Arg Lys, OrnithineAsn Gln Asp Glu Glu Asp Gln Asn Gly Ala Ile Val, Leu, Met, Nle Leu Ile,Val, Met, Nle Lys Arg, Ornithine Met Leu, Ile, Nle, Val Ornithine Lys,Arg Phe Tyr, Trp Ser Thr Thr Ser Trp Phe, Tyr Tyr Phe, Trp Val Leu, Ile,Met, Nle

Thus, for example, the following oligopeptides may be synthesized bytechniques well known to persons of ordinary skill in the art and wouldbe expected to be proteolytically cleaved by free PSA:

AsnArgIleSerTyrGln|Ser (SEQ.ID.NO.: 49)

AsnLysValSerTyrGln|Ser (SEQ.ID.NO.: 50)

AsnLysMetSerTyrGln|SerSer (SEQ.ID.NO.: 51)

AsnLysLeuSerTyrGln|SerSer (SEQ.ID.NO.: 52)

AsnLysIleSerTyrGln|Ser (SEQ.ID.NO.: 53)

GlnLysIleSerTyrGln|SerSer (SEQ.ID.NO.: 54).

The inclusion of the symbol “|” within an amino acid sequence indicatesthe point within that sequence where the oligopeptide is proteolyticallycleaved by free PSA.

The compounds of the present invention may have asymmetric centers andoccur as racemates, racemic mixtures, and as individual diastereomers,with all possible isomers, including optical isomers, being included inthe present invention. Unless otherwise specified, named amino acids areunderstood to have the natural “L” stereoconfiguration

The following abbreviations are utilized in the specification andfigures to denote the indicated amino acids and moieties:

hR or hArg: homoarginine hY or hTyr: homotyrosine Cha: cyclohexylalanineAmf: 4-aminomethylphenylalanine DPL: 2-(4,6-dimethylpyrimidinyl)lysine(imidazolyl)K: N′-(2-imidazolyl)lysine Me₂PO₃-Y:O-dimethylphosphotyrosine O-Me-Y: O-methyltyrosine TIC:tetrahydro-3-isoquinoline carboxylic acid DAP: 1,3-diaminopropane TFA:trifluoroacetic acid AA: acetic acid 3PAL 3-pyridyl-alanine

The conjugates of the instant invention comprise oligomers wherein theN-terminus amino acid is modified with a hydrophilic blocking group.Such blocking groups are chosen based upon the presence of hydrophilicfunctionality. The presence of the hydrophilic functionalitydistinguishes the instant conjugates from conjugates previouslydisclosed that also had N-terminus blocking groups. Such blocking of theterminal amino group may also reduce or eliminate the enzymaticdegradation of such peptidyl therapeutic agents by the action ofexogenous amino peptidases which are present in the blood plasma of warmblooded animals. Blocking groups that increase the hydrophilicity of theconjugates and therefore increase the aqueous solubilily of theconjugates include but are not limited to hydroylated alkanoyl,polyhydroxylated alkanoyl, polyethylene glycol, glycosylates, sugars andcrown ethers.

Preferably the blocking group is selected from

wherein:

R¹ and R² are independently selected from:

a) hydrogen,

b) unsubstituted or substituted aryl, unsubstituted or substitutedheterocycle, C₃-C₁₀ cycloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, halogen,C₁-C₆ perfluoroalkyl, R¹²O—, R³C(O)NR³—, (R³)₂NC(O)—, R³ ₂N—C(NR³)—,R⁴S(O)_(m)NH, CN, NO₂, R³C(O)—, N₃, —N(R³)₂, or R⁴OC(O)NR³—,

c) unsubstituted C₁-C₆ alkyl,

d) substituted C₁-C₆ alkyl wherein the substituent on the substitutedC₁-C₆ alkyl is selected from unsubstituted or substituted aryl,unsubstituted or substituted heterocyclic, C₃-C₁₀ cycloalkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, R³O—, R⁴S(O)_(m)NH, R³C(O)NR³—, (R³)₂NC(O)—, R³₂N—C(NR³)—, CN, R³C(O)—, N₃, —N(R³)₂, and R⁴OC(O)—NR³—; or

R¹ and R² are combined to form —(CH₂)_(s)— wherein one of the carbonatoms is optionally replaced by a moiety selected from: O, S(O)_(m),—NC(O)—, NH and —N(COR⁴)—;

R³ is selected from: hydrogen, aryl, substituted aryl heterocycle,substituted heterocycle, C₁-C₆ alkyl and C₃-C₁₀ cycloalkyl;

R⁴ is selected from: aryl, substituted aryl, heterocycle, substitutedheterocycle, C₁-C₆ alkyl and C₃-C₁₀ cycloalkyl;

m is 0, 1 or 2;

n is 1, 2, 3 or 4;

p is zero or an integer between 1 and 100; and

q is 0 or 1, provided that if p is zero, q is 1; and

s is 3, 4 or 5.

The conjugates of the present invention may have asymmetric centers andoccur as racemates, racemic mixtures, and as individual diastereomers,with all possible isomers, including optical isomers, being included inthe present invention. When any variable (e.g. aryl, heterocycle, R³etc.) occurs more than one time in any constituent, its definition oneach occurence is independent of every other occurence. For example,HO(CR¹R²)₂— represents HOCH₂CH₂—, HOCH₂CH(OH)—, HOCH(CH₃)CH(OH)—, etc.Also, combinations of substituents and/or variables are permissible onlyif such combinations result in stable compounds.

As used herein, “alkyl” and the alkyl portion of aralkyl and similarterms, is intended to include both branched and straight-chain saturatedaliphatic hydrocarbon groups having the specified number of carbonatoms; “alkoxy” represents an alkyl group of indicated number of carbonatoms attached through an oxygen bridge.

As used herein, “cycloalkyl” is intended to include non-aromatic cyclichydrocarbon groups having the specified number of carbon atoms. Examplesof cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl and the like.

“Alkenyl” groups include those groups having the specified number ofcarbon atoms and having one or several double bonds. Examples of alkenylgroups include vinyl, allyl, isopropenyl, pentenyl, hexenyl, heptenyl,cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, 1-propenyl,2-butenyl, 2-methyl-2-butenyl, isoprenyl, farnesyl, geranyl,geranylgeranyl and the like.

“Alkynyl” groups include those groups having the specified number ofcarbon atoms and having one triple bonds. Examples of alkynyl groupsinclude acetylene, 2-butynyl, 2-pentynyl, 3-pentynyl and the like.

“Halogen” or “halo” as used herein means fluoro, chloro, bromo and iodo.

As used herein, “aryl,” and the aryl portion of aralkyl and aroyl, isintended to mean any stable monocyclic or bicyclic carbon ring of up to7 members in each ring, wherein at least one ring is aromatic. Examplesof such aryl elements include phenyl, naphthyl, tetrahydronaphthyl,indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl.

The term heterocycle or heterocyclic, as used herein, represents astable 5- to 7-membered monocyclic or stable 8- to 11-membered bicyclicheterocyclic ring which is either saturated or unsaturated, and whichconsists of carbon atoms and from one to four heteroatoms selected fromthe group consisting of N, O, and S, and including any bicyclic group inwhich any of the above-defined heterocyclic rings is fused to a benzenering. The heterocyclic ring may be attached at any heteroatom or carbonatom which results in the creation of a stable structure. Examples ofsuch heterocyclic elements include, but are not limited to, azepinyl,benzimidazolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl,benzothiopyranyl, benzofuryl, benzothiazolyl, benzothienyl,benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl,dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranylsulfone, furyl, imidazolidinyl, imidazolinyl, imidazolyl, indolinyl,indolyl, isochromanyl, isoindolinyl, isoquinolinyl, isothiazolidinyl,isothiazolyl, isothiazolidinyl, morpholinyl, naphthyridinyl,oxadiazolyl, 2-oxoazepinyl, oxazolyl, 2-oxopiperazinyl, 2-oxopiperdinyl,2-oxopyrrolidinyl, piperidyl, piperazinyl, pyridyl, pyrazinyl,pyrazolidinyl, pyrazolyl, pyridazinyl, pyrimidinyl, pyrrolidinyl,pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrahydrofuryl,tetrahydroisoquinolinyl, tetrahydroquinolinyl, thiamorpholinyl,thiamorpholinyl sulfoxide, thiazolyl, thiazolinyl, thienofuryl,thienothienyl, and thienyl.

As used herein in the terms “substituted C₁₋₈ alkyl”, “substituted aryl”and “substituted heterocycle” include moieties containing from 1 to 3substituents in addition to the point of attachment to the rest of thecompound. Such additional substituents are selected from F, Cl, Br, CF₃,NH₂, N(C₁-C₆ alkyl)₂, NO₂, CN, (C₁-C₆ alkyl)O—, —OH, (C₁-C₆alkyl)S(O)_(m)—, (C₁-C₆ alkyl)C(O)NH—, H₂N—C(NH)—, (C₁-C₆ alkyl)C(O)—,(C₁-C₆ alkyl)OC(O)—, N₃, (C₁-C₆ alkyl)OC(O)NH— and C₁-C₂₀ alkyl.

When R¹ and R² are combined to form —(CH₂)_(s)—, the cyclic moieties andheteroatom-containing cyclic moieties so defined include, but are notlimited to:

As used herein, the term “PEG” represents certain polyethylene glycolcontaining substituents having the designated number of ethyleneoxysubunits. Thus the term PEG(2) represents

and the term PEG(6) represents

As used herein, the term “(d)(2,3-dihydroxypropionyl)” represents thefollowing structure:

As used herein, the term “(2R,3S) 2,3,4-trihydroxybutanoyl” representsthe following structure:

Because the conjugates of the invention can be used for modifying agiven biological response, cytotoxic agent is not to be construed aslimited to classical chemical therapeutic agents. For example, thecytotoxic agent may be a protein or polypeptide possessing a desiredbiological activity. Such proteins may include, for example, a toxinsuch as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; aprotein such as tumor necrosis factor, α-interferon, β-interferon, nervegrowth factor, platelet derived growth factor, tissue plasminogenactivator; or, biological response modifiers such as, for example,lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

The preferred cytotoxic agents include, in general, alkylating agents,antiproliferative agents, tubulin binding agents and the like. Preferredclasses of cytotoxic agents include, for example, the anthracyclinefamily of drugs, the vinca drugs, the mitomycins, the bleomycins, thecytotoxic nucleosides, the taxanes, the pteridine family of drugs,diynenes and the podophyllotoxins. Particularly useful members of thoseclasses include, for example, doxorubicin, carminomycin, daunorubicin,aminopterin, methotrexate, methopterin, dichloro-methotrexate, mitomycinC, porfiromycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside,podophyllotoxin, or podophyllotoxin derivatives such as etoposide oretoposide phosphate, melphalan, vinblastine, vincristine, leurosidine,vindesine, leurosine, taxol and the like. Other useful cytotoxic agentsinclude estramustine, cisplatin and cyclophosphamide. One skilled in theart may make chemical modifications to the desired cytotoxic agent inorder to make reactions of that compound more convenient for purposes ofpreparing conjugates of the invention.

A highly preferred group of cytotoxic agents for the present inventioninclude drugs of the following formulae:

The Methotrexate Group of Formula(1)

in which

R¹² is amino or hydroxy;

R⁷ is hydrogen or methyl;

R⁸ is hydrogen, fluoro, chloro, bromo or iodo;

R⁹ is hydroxy or a moiety which completes a salt of the carboxylic acid;

The Mitomycin Group of Formula (2)

in which

R¹⁰ is hydrogen or methyl;

The Bleomycin Group of Formula (3)

in which R¹¹ is hydroxy, amino, C₁-C₃ alkylamino, di(C₁-C₃ alkyl)amino,C₄-C₆ polymethylene amino,

Melphalan of Formula (4)

6-Mercaptopurine of Formula (5)

A Cytosine Arabinoside of Formula (6)

The Podophyllotoxins of Formula(7)

in which

R¹³ is hydrogen or methyl;

R¹⁴ is methyl or thienyl;

or a phosphate salt thereof;

The Vinca Alkaloid Group of Drugs of Formula (8)

in which

R¹⁵ is H, CH₃ or CHO; when R¹⁷ and R¹⁸ are taken singly;

R¹⁸ is H, and one of R¹⁶ and R¹⁷ is ethyl and the other is H or OH; whenR¹⁷ and R¹⁸ are taken together with the carbons to which they areattached, they form an oxirane ring in which case R¹⁶ is ethyl;

R¹⁹ is hydrogen, (C₁-C₃ alkyl)—CO, or chlorosubstituted (C₁-C₃alkyl)—CO;

Difluornucleosides of Formula (9)

in which

R²¹ is a base of one of the formulae:

 in which

R²² is hydrogen, methyl, bromo, fluoro, chloro or iodo;

R²³ is —OH or —NH₂;

R²⁴ is hydrogen, bromo, chloro or iodo; or,

The Anthracyclines Antibiotics of Formula (10)

wherein

R^(a) is —CH₃, —CH₂OH, —CH₂OCO(CH₂)₃CH₃, or —CH₂OCOCH(OC₂H₅)₂;

R^(b) is —OCH₃, —OH or —H;

R^(c) is —NH₂, —NHCOCF₃, 4-morpholinyl, 3-cyano-4-morpholinyl,1-piperidinyl, 4-methoxy-1-piperidinyl, benzylamine, dibenzylamine,cyanomethylamine, or 1-cyano-2-methoxyethyl amine;

R5 is —OH —OTHP or —H; and

R⁶ is —OH or —H provided that R⁶ is not —OH when R⁵ is —OH or —OTHP.

Estramustine (11)

Cyclophosphamide (12)

The most highly preferred drugs are the anthracycline antiobiotic agentsof Formula (10), described previously. One skilled in the artunderstands that this structural formula includes compounds which aredrugs, or are derivatives of drugs, which have acquired in the artdifferent generic or trivial names. Table 1, which follows, represents anumber of anthracycline drugs and their generic or trivial names andwhich are especially preferred for use in the present invention.

TABLE 1 (11)

Com- pound R^(a) R^(b) R^(c) R₅ R₆ dauno- CH₃ OCH₃ NH₂ OH H rubicin^(a)doxo- CH₂OH OCH₃ NH₂ OH H rubicin^(b) deto- CH₂OCOCH(OC₂H₅)₂ OCH₃ NH₂ OHH rubicin carmin- CH₃ OH NH₂ OH H omycin ida- CH₃ H NH₂ OH H rubicinepi- CH₂OH OCH₃ NH₂ OH OH rubicin eso- CH₂OH OCH₃ NH₂ H H rubicin THPCH₂OH OCH₃ NH₂ OTHP H AD-32 CH₂OCO(CH₂)₃CH₃ OCH₃ NHCOCF₃ OH H^(a)“daunomycin” is an alternative name for daunorubicin^(b)“adriamycin” is an alternative name for doxorubicin

Of the compounds shown in Table 1, the most highly preferred cytotoxicagents are doxorubicin, vinblastine and desacetylvinblastine.Doxorubicin (also referred to herein as “DOX”) is that anthracycline ofFormula (10) in which R^(a) is —CH₂OH, R^(c) is —OCH₃, R⁴ is —NH₂, R⁵ is—OH, and R⁶ is —H.

The blocked oligopeptide-cytotoxic agent conjugate of the instantinvention wherein the cytotoxic agent is the preferred cytotoxic agentdoxorubicin may be described by the general formula I below:

wherein:

oligopeptide is an oligopeptide which is selectively recognized by thefree prostate specific antigen (PSA) and is capable of beingproteolytically cleaved by the enzymatic activity of the free prostatespecific antigen, and wherein the C-terminus carbonyl is covalentlybound to the amine of doxorubicin and the N-terminus amine is covalentlybound to the carbonyl of the blocking group;

R is selected from

R¹ and R² are independently selected from: hydrogen, OH, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ aralkyl and aryl;

n is 1, 2, 3 or 4;

p is zero or an integer between 1 and 100;

q is 0 or 1, provided that if p is zero, q is 1;

or the pharmaceutically acceptable salt thereof.

In a preferred embodiment of the oligopeptide-cytotoxic agent conjugate:

R is selected from

R¹ and R² are independently selected from: hydrogen, C₁-C₆ alkyl andaryl;

n is 1, 2, 3 or 4;

n is 0, 1, 2 or 3;

p is zero or an integer between 1 and 14;

q is 0 or 1, provided that if p is zero, q is 1;

or the pharmaceutically acceptable salt thereof.

The following compounds are specific examples of theoligopeptide-cytotoxic agent conjugate of the instant invention:

wherein X is:

or the pharmaceutically acceptable salt thereof.

Further examples of conjugates of an oligopeptide and doxorubicinwherein the N-terminus of the oligopeptide is blocked by a hydrophilicmoiety and the C-terminus of the oligopeptide is attached to thedoxorubicin at the 3′-amine are as follows:

2-hydroxyacetyl-hArgSerSerTyrGln-SerNle-DOX (3′) (SEQ.ID.NO.: 64)

2-hydroxyacetyl-hArgSerSerChgGln-SerNle-DOX (3′) (SEQ.ID.NO.: 65)

2-hydroxyacetyl-SerhArgChgGln-SerLeu-DOX (3′) (SEQ.ID.NO.: 66)

2-hydroxyacetyl-hArgSerSerChgGln-SerLeu-DOX (3′) (SEQ.ID.NO.: 67)

2-hydroxyacetyl-hArgAlaSerChgGln-SerLeu-DOX (3′) (SEQ.ID.NO.: 68)

(d) 2,3-dihydroxypropionyl-SerhArgChgGln-SerLeu-DOX (3′) (SEQ.ID.NO.:69)

(l) 2,3-dihydroxypropionyl-SerhArgChgGln-SerLeu-DOX (3′) (SEQ.ID.NO.:70)

PEG(2)-SerhArgChgGln-SerLeu-DOX (3′) (SEQ.ID.NO.: 71)

PEG(2)-hArgChgGln-SerLeu-DOX (3′) (SEQ.ID.NO.: 72)

(2R,3S) 2,3,4-trihydroxybutanoyl-hArgChgGln-SerLeu-DOX (3′) (SEQ.ID.NO.:73)

PEG(2)-SerhArgTyrGln-SerLeu-DOX(3′) (SEQ.ID.NO.: 74)

PEG(2)-hArgTyrGln-SerSerSerLeu-DOX (3′) (SEQ.ID.NO.: 75)

PEG(2)-LysTyrGln-SerSerSerLeu-DOX (3′) (SEQ.ID.NO.: 76)

2-hydroxyacetyl-hArgSerSerTyrGln-SerLeu-DOX (3′) (SEQ.ID.NO.: 77)

(l)(2,3-dihydroxypropionyl)hArgSerSerChgGlnSerLeu-DOX (3′) (SEQ.ID.NO.:78)

PEG(2)-hArgSerSerChgGln-SerLeu-DOX (3′) (SEQ.ID.NO.: 79)

2-hydroxyacetyl-SerTyrGln-SerSerSerLeu-DOX (3′) (SEQ.ID.NO.: 80)

PEG(16)-SerhArgTyrGln-SerLeu-DOX (3′) (SEQ.ID.NO.: 81)

(2R,3S) 2,3,4-trihydroxybutanoyl-SerhArgChgGln-SerLeu-DOX (3′)(SEQ.ID.NO.: 82)

PEG(2)-SerhArgTyrGln-SerLeu-DOX (3′) (SEQ.ID.NO.: 83)

(d)(2,3-dihydroxypropionyl)-hArgSerSerChgGln-SerLeu-DOX(3′) (SEQ.ID.NO.:84)

(l)(2,3-dihydroxypropionyl)SerSerSerChgGln-Ser(dLeu)-DOX (3′)(SEQ.ID.NO.: 85)

(d)(2,3-dihydroxypropionyl)SerSerSerChgGln-SerLeu-DOX (3′) (SEQ.ID.NO.:86)

(l)(2,3-dihydroxypropionyl)SerSerSerChgGln-SerLeu-DOX (3′) (SEQ.ID.NO.:87)

(l)(2,3-dihydroxypropionyl)SerSerChgGln-Ser(dLeu)-DOX (3′) (SEQ.ID.NO.:88)

(d)(2,3-dihydroxypropionyl)SerSerChgGln-SerLeu-DOX (3′) (SEQ.ID.NO.: 89)

PEG(2)-SerSerChgGln-Ser(dLeu)-DOX (3′) (SEQ.ID.NO.: 90)

PEG(2)SerSerChgGln-SerLeu-DOX (3′) (SEQ.ID.NO.: 91)

PEG(2)-SerSerSerChgGln-Ser(dLeu)-DOX (3′) (SEQ.ID.NO.: 92)

(2,3-dihydroxypropionyl)-3PAL-SerSerChgGln-Ser(dLeu)-DOX (3′)(SEQ.ID.NO.: 93)

(d)(2,3-dihydroxypropionyl)-3PAL-SerSerChgGln-SerLeu-DOX (3′)(SEQ.ID.NO.: 94)

(l)(2,3-dihydroxypropionyl)-SerSerChgGln-SerLeu-DOX (3′) (SEQ.ID.NO.:95)

(2,3-dihydroxypropionyl)-hSerSerSerChgGln-SerLeu-DOX (3′) (SEQ.ID.NO.:96)

PEG(2)-AlaSerChgGln-SerLeu-DOX (3′) (SEQ.ID.NO.: 97)

PEG(6)-SerSerChgGln-SerLeu-DOX (3′) (SEQ.ID.NO.: 98)

PEG(6)-SerSerSerChgGln-SerLeu-DOX (3′) (SEQ.ID.NO.: 99)

PEG(6)-AlaSerChgGln-SerLeu-DOX (3′) (SEQ.ID.NO.: 100)

PEG(4)-3PALSerSerChgGln-SerLeu-DOX (3′) (SEQ.ID.NO.: 101)

or the pharmaceutically acceptable salt thereof.

The oligopeptide-cytotoxic agent conjugate of the instant inventionwherein the cytotoxic agent is the preferred cytotoxic agent vinblastineor desacetylvinblastine may be described by the general formula IIbelow:

wherein:

oligopeptide is an oligopeptide which is specifically recognized by thefree prostate specific antigen (PSA) and is capable of beingproteolytically cleaved by the enzymatic activity of the free prostatespecific antigen;

X_(L) is —NH—(CH₂)_(r)—NH—

R is selected from

R¹ and R² are independently selected from: hydrogen, OH, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ aralkyl and aryl;

R¹⁹ is hydrogen, (C₁-C₃ alkyl)—CO, or chlorosubstituted (C₁-C₃alkyl)—CO;

n is 1, 2, 3 or 4;

p is zero or an integer between 1 and 100;

q is 0 or 1, provided that if p is zero, q is 1;

r is 1, 2, 3, 4 or 5,

or the pharmaceutically acceptable salt thereof.

The another embodiment of the oligopeptide-cytotoxic agent conjugate ofthe instant invention wherein the cytotoxic agent is the preferredcytotoxic agent vinblastine or desacetylvinblastine may be described bythe general formula III below:

wherein:

oligopeptide is an oligopeptide which is specifically recognized by thefree prostate specific antigen (PSA) and is capable of beingproteolytically cleaved by the enzymatic activity of the free prostatespecific antigen,

R^(d) and R^(e) are independently selected from: hydrogen, C₁-C₆-alkyl,—C₁-C₆-alkyl-OH, —C₁-C₆-alkyl-di-OH, —C₁-C₆-alkyl-tri-OH and

 provided that at least one R^(d) and R^(e) are not hydrogen orC₁-C₆-alkyl, or

R^(d) and R^(e) are combined to form a —CH₂CH₂OCH₂CH₂—diradical;

R¹⁹ is hydrogen, (C₁-C₃ alkyl)—CO, or chlorosubstituted (C₁-C₃alkyl)—CO;

p is zero or an integer between 1 and 100;

q is 0 or 1, provided that if p is zero, q is 1;

The following compounds are specific examples of theoligopeptide-desacetylvinblastine conjugate of the instant invention:

or the pharmaceutically acceptable salt thereof.

The oligopeptides, peptide subunits and peptide derivatives (also termed“peptides”) of the present invention can be synthesized from theirconstituent amino acids by conventional peptide synthesis techniques,preferably by solid-phase technology. The peptides are then purified byreverse-phase high performance liquid chromatography (HPLC).

Standard methods of peptide synthesis are disclosed, for example, in thefollowing works: Schroeder et al., “The Peptides”, Vol. I, AcademicPress 1965; Bodansky et al., “Peptide Synthesis”, IntersciencePublishers, 1966; McOmie (ed.) “Protective Groups in Organic Chemistry”,Plenum Press, 1973; Barany et al., “The Peptides: Analysis, Synthesis,Biology” 2, Chapter 1, Academic Press, 1980, and Stewart et al., “SolidPhase Peptide Synthesis”, Second Edition, Pierce Chemical Company, 1984.The teachings of these works are hereby incorporated by reference.

The pharmaceutically acceptable salts of the compounds of this inventioninclude the conventional non-toxic salts of the compounds of thisinvention as formed, e.g., from non-toxic inorganic or organic acids.For example, such conventional non-toxic salts include those derivedfrom inorganic acids such as hydrochloric, hydrobromic, sulfuric,sulfamic, phosphoric, nitric and the like: and the salts prepared fromorganic acids such as acetic, propionic, succinic, glycolic, stearic,lactic, malic, tartaric, citric, ascorbic, pamoic, maleic,hydroxymaleic, phenyl-acetic, glutamic, benzoic, salicylic, sulfanilic,2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, trifluoroacetic and the like.

The conjugates of the instant invention which comprise the oligopeptidecontaining the PSA cleavage site and a cytotoxic agent may similarly besynthesized by techniques well known in the medicinal chemistry art. Forexample, a free amine moiety on the cytotoxic agent may be covalentlyattached to the oligopeptide at the carboxyl terminus such that an amidebond is formed. Similarly, an amide bond may be formed by covalentlycoupling an amine moiety of the oligopeptide and a carboxyl moiety ofthe cytotoxic agent. For these purposes a reagent such as2-(1H-benzotriazol-1-yl)-1,3,3-tetramethyluronium hexafluorophosphate(known as HBTU) and 1-hyroxybenzotriazole hydrate (known as HOBT),dicyclohexyl-carbodiimide (DCC),N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide (EDC),diphenylphosphorylazide (DPPA),benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphoniumhexafluorophosphate (BOP) and the like, used in combination orsingularly, may be utilized.

Furthermore, the instant conjugate may be formed by a non-peptidyl bondbetween the PSA cleavage site and a cytotoxic agent. For example, thecytotoxic agent may be covalently attached to the carboxyl terminus ofthe oligopeptide via a hydroxyl moiety on the cytotoxic agent, therebyforming an ester linkage. For this purpose a reagent such as acombination of HBTU and HOBT, a combination of BOP and imidazole, acombination of DCC and DMAP, and the like may be utilized. Thecarboxylic acid may also be activated by forming the nitro-phenyl esteror the like and reacted in the presence of DBU(1,8-diazabicyclo[5,4,0]undec-7-ene.

The instant conjugate may also be formed by attachment of theoligopeptide to the cytotoxic agent via a linker unit. Such linker unitsinclude, for example, a biscarbonyl alkyl diradical whereby an aminemoiety on the cytotoxic agent is connected with the linker unit to forman amide bond and the amino terminus of the oligopeptide is connectedwith the other end of the linker unit also forming an amide bond.Conversely, a diaminoalkyl diradical linker unit, whereby a carbonylmoiety on the cyctotoxic agent is covalently attacted to one of theamines of the linker unit while the other amine of the linker unit iscovalently attached to the C terminus of the oligopeptide, may also beuselful. Other such linker units which are stable to the physiologicalenvironment when not in the presence of free PSA, but are cleavable uponthe cleavage of the PSA proteolytic cleavage site, are also envisioned.Furthermore, linker units may be utilized that, upon cleavage of the PSAproteolytic cleavage site, remain attached to the cytotoxic agent but donot significantly decrease the cytotoxic activity of such apost-cleavage cytotoxic agent derivative when compared with anunmodified cytotoxic agent.

One skilled in the art understands that in the synthesis of compounds ofthe invention, one may need to protect various reactive functionalitieson the starting compounds and intermediates while a desired reaction iscarried out on other portions of the molecule. After the desiredreactions are complete, or at any desired time, normally such protectinggroups will be removed by, for example, hydrolytic or hydrogenolyticmeans. Such protection and deprotection steps are conventional inorganic chemistry. One skilled in the art is referred to ProtectiveGroups in Organic Chemistry, McOmie, ed., Plenum Press, NY, N.Y. (1973);and, Protective Groups in Organic Svnthesis, Greene, ed., John Wiley &Sons, NY, N.Y. (1981) for the teaching of protective groups which may beuseful in the preparation of compounds of the present invention.

By way of example only, useful amino-protecting groups may include, forexample, C₁-C₁₀ alkanoyl groups such as formyl, acetyl, dichloroacetyl,propionyl, hexanoyl, 3,3-diethylhexanoyl, γ-chlorobutryl, and the like;C₁-C₁₀ alkoxycarbonyl and C₅-C₁₅ aryloxycarbonyl groups such astert-butoxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl,4-nitrobenzyloxycarbonyl, fluorenylmethyloxycarbonyl andcinnamoyloxycarbonyl; halo-(C₁-C₁₀)-alkoxycarbonyl such as2,2,2-trichloroethoxycarbonyl; and C₁-C₁₅ arylalkyl and alkenyl groupsuch as benzyl, phenethyl, allyl, trityl, and the like. Other commonlyused amino-protecting groups are those in the form of enamines preparedwith β-keto-esters such as methyl or ethyl acetoacetate.

Useful carboxy-protecting groups may include, for example, C₁-C₁₀ alkylgroups such as methyl, tert-butyl, decyl; halo-C₁-C₁₀ alkyl such as2,2,2-trichloroethyl, and 2-iodoethyl; C₅-C₁₅ arylalkyl such as benzyl,4-methoxybenzyl, 4-nitrobenzyl, triphenylmethyl, diphenyl-methyl; C₁-C₁₀alkanoyloxymethyl such as acetoxy-methyl, propionoxymethyl and the like;and groups such as phenacyl, 4-halophenacyl, allyl, dimethylallyl,tri-(C₁-C₃ alkyl)silyl, such as trimethylsilyl,β-p-toluenesulfonylethyl, β-p-nitrophenyl-thioethyl,2,4,6-trimethylbenzyl, β-methylthioethyl, phthalimidomethyl,2,4-dinitro-phenylsulphenyl, 2-nitrobenzhydryl and related groups.

Similarly, useful hydroxy protecting groups may include, for example,the formyl group, the chloroacetyl group, the benzyl group, thebenzhydryl group, the trityl group, the 4-nitrobenzyl group, thetrimethylsilyl group, the phenacyl group, the tert-butyl group, themethoxymethyl group, the tetrahydropyranyl group, and the like.

With respect to the preferred embodiment of an oligopeptide combinedwith the anthracycline antibiotic doxorubicin, the following ReactionSchemes illustrate the synthesis of the conjugates of the instantinvention.

Reaction Scheme VI illustrates preparation of conjugates of theoligopeptides of the instant invention and the vinca alkaloid cytotoxicagent vinblastine wherein the attachment of vinblastine is at theC-terminus of the oligopeptide. The use of the 1,3-diaminopropane linkeris illustrative only; other spacer units between the carbonyl ofvinblastine and the C-terminus of the oligopeptide are also envisioned.Furthermore, Scheme VI illustrates a synthesis of conjugates wherein theC-4-position hydroxy moiety is reacetylated following the addition ofthe linker unit. Applicants have discovered that the desacetylvinblastine conjugate is also efficacious and may be prepared byeliminating the steps shown in Reaction Scheme VI of protecting theprimary amine of the linker and reacting the intermediate with aceticanhydride, followed by deprotection of the amine. Conjugation of theoligopeptide at other positions and functional groups of vinblastine maybe readily accomplished by one of ordinary skill in the art and is alsoexpected to provide compounds useful in the treatment of prostatecancer.

The oligopeptide-cytotoxic agent conjugates of the invention areadministered to the patient in the form of a pharmaceutical compositionwhich comprises a conjugate of of the instant invention and apharmaceutically acceptable carrier, excipient or diluent therefor. Asused, “pharmaceutically acceptable” refers to those agents which areuseful in the treatment or diagnosis of a warm-blooded animal including,for example, a human, equine, procine, bovine, murine, canine, feline,or other mammal, as well as an avian or other warm-blooded animal. Thepreferred mode of administration is parenterally, particularly by theintravenous, intramuscular, subcutaneous, intraperitoneal, orintralymphatic route. Such formulations can be prepared using carriers,diluents or excipients familiar to one skilled in the art. In thisregard, See, e.g. Remington's Pharmaceutical Sciences, 16th ed., 1980,Mack Publishing Company, edited by Osol et al. Such compositions mayinclude proteins, such as serum proteins, for example, human serumalbumin, buffers or buffering substances such as phosphates, othersalts, or electrolytes, and the like. Suitable diluents may include, forexample, sterile water, isotonic saline, dilute aqueous dextrose, apolyhydric alcohol or mixtures of such alcohols, for example, glycerin,propylene glycol, polyethylene glycol and the like. The compositions maycontain preservatives such as phenethyl alcohol, methyl and propylparabens, thimerosal, and the like. If desired, the composition caninclude about 0.05 to about 0.20 percent by weight of an antioxidantsuch as sodium metabisulfite or sodium bisulfite.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specific amounts, aswell as any product which results, directly or indirectly, fromcombination of the specific ingredients in the specified amounts.

For intravenous administration, the composition preferably will beprepared so that the amount administered to the patient will be fromabout 0.01 to about 1 g of the conjugate. Preferably, the amountadministered will be in the range of about 0.2 g to about 1 g of theconjugate. The conjugates of the invention are effective over a widedosage range depending on factors such as the disease state to betreated or the biological effect to be modified, the manner in which theconjugate is administered, the age, weight and condition of the patientas well as other factors to be determined by the treating physician.Thus, the amount administered to any given patient must be determined onan individual basis.

One skilled in the art will appreciate that although specific reagentsand reaction conditions are outlined in the following examples,modification can be made which are meant to be encompassed by the spiritand scope of the invention. The following preparations and examples,therefore, are provided to further illustrate the invention, and are notlimiting.

EXAMPLES Example 1

Preparation of Oligopeptides which Comprise the PSA Mediated CleavageSite

Blocked oligopeptides were prepared by solid-phase synthesis, using adouble coupling protocol for the introduction of amino acids on theApplied Biosystems model 430A automated peptide synthesizer.Deprotection and removal of the oligopeptide from the resin support wereachieved by treatment with liquid hydrofluoric acid. The oligopeptideswere purified by preparative high pressure liquid chromatography onreverse phase C18 silica columns using an aqueous 0.1% trifluoroaceticacid/acetonitrile gradient. Identity and homogeneity of theoligopeptides were confirmed by amino acid composition analysis, highpressure liquid chromatography, and fast atom bombardment mass spectralanalysis. The oligopeptides that were prepared by this method are shownin Table 2.

TABLE 2 SEQ. Time to 50% Substrate ID. Cleavage by NO.PEPTIDE/PEPTIDE-DOX CONJUGATE York PSA (Min) 103 Ac-ANKASYQ-SL-acid 135104 Ac-ANKASYQ-SL-acid 220 105 Ac-hR(CHA)Q-SNle-acid 200 (PS) 106Ac-ShRYQ-SNle-acid  25 (PS) 107 AG-ShRChgQ-SNle-acid INSOLUBLE 108Ac-hRSSYQ-SNle-acid  25 (PS) 109 AchRSSChgQ-SL-acid 120 (45*)  662-hydroxyacetyl-ShRChgQ-SL-acid 120 (30*) 110 Ac-hRSSYQ-SNle-acid  25(PS)  64 2-hydroxyacetyl-hRSSYQ-SNle-acid  45 111 Ac-hRASChgQ-SL-acid 50  68 2-hydroxyacetyl-hRASChgQ-SL-acid  70  642-hydroxyacetyl-hRSSYQ-SL-acid  35 (PP)  672-hydroxyacetyl-hRSSChgSL-acid (PP)  692,3-dihydroxypropionyl-ShRChgQ-SL-  75 acid  702(S)-2,3-dihydroxy-propionyl  35* ShRChgQSL-acid 1122-hydroxyacetylhRYQ-SL-acid 105*  29 ShRChgQ-SL-acid 4 HOUR = 8%  71PEG(2)-S-hRChgQ-SL-acid  30 113 PEG(1)-ShRChgQ-SL-acid 120*  782(S)2,3-dihydroxypropionyl-hRSSChgQ-  25* SL-acid  79PEG(2)-hRSSChgQ-SL-acid  40*  74 PEG(2)-ShRYQ-SL-acid  35* 114PEG(1)-hRSSChgQ-SL-acid  30 115 PEG(1)-ShRYQ-SL-acid  90 116PEG(15)-ShRYQ-SL-acid  40  81 PEG(16)-ShRYQ-SL-acid  40 117PEG(17)-ShRYQ-SL-acid  55  82 (2R,3S) 2,3,4-trihydroxybutanoyl-  90ShRChgQ-SL-acid  83 2,3-dihydroxypropionyl-hRSSChgQ-SL-  50 acid 118PEG(2)-SSYQ-SL-acid 150 119 PEG(14)ShRYQ-SL-acid  40 120PEG(18)ShRYQ-SL-acid  40 121 PEG(19)ShRYQ-SL-acid  60  94(d)2,3-dihydroxypropionyl-3PAL-  80 SSChgQSL-acid  63PEG(2)SSSChgQ-SL-acid 150 101 PEG(2)-3PAL-SSChgQ-SL-acid  80  87(l)2,3-dihydroxypropionyl-SSSChgQ-SL-  80 acid  61(OH-Ac)SSSChgQ-SL-acid 120 122 (l)2,3-dihydroxypropionyl-SSChgQ-SL- 180acid  87 (l)2,3-dihydroxypropionyl-SSSChgQ-SL- 110 acid 123(l)2,3-dihydroxypropionyl-3PAL-  70 SSChgQ-SL-acid 1242,3-dihydroxypropionyl-SSSChgQ-SL- 120 acid 1252,3-dihydroxypropionyl-ShRYQ-SL-  35 acid  622-hydroxyacetyl-SSChgQ-SL-acid 180 1252,3-dihydroxypropionyl-ShRYQ-SL-acid  50 101 PEG(4)-β-PAL-SSChgQ-SL-acid 60 126 Ac-SSSChgQ-SV-acid 127 Ac-PSSChgQ-SV-acid 1282,3-dihydroxypropionyl-GSSChgQ-SL- 160 acid  962,3-dihydroxypropionyl-hSSSChgQ-SL- 160 acid

Example 2

Assessment of the Recognition of Oligopeptides by Free PSA

The oligopeptides prepared as described in Example 1 were individuallydissolved in PSA digestion buffer (12 mMtris(hydroxymethyl)-aminomethane pH8.0, 25 mM NaCl, 0.5 mM CaCl₂) andthe solution added to PSA at a molar ration of 100 to 1. The reaction isquenched after various reaction times by the addition of trifluoroaceticacid (TFA) to a final 1% (volume/volume). The quenched reaction wasanalyzed by HPLC on a reversed-phase C18 column using an aqueous 0.1%TFA/acetonitrile gradient. The results of the assessment are shown inTable 2. Table 2 shows the amount of time (in minutes) required for 50%cleavage of the noted oligopeptides with enzymatically active free PSA.Oligopeptides containing free amine moieties (ie. comprising hArg, Orn,Lys and or 3PAL) were tested as TFA salts. All other oligopeptides weretested as neutral compounds.

Example 3

Preparation of N-(2-Hydroxyacetyl)-Ser-Ser-Ser-Chg-Gln-Ser-Leu-Dox (3-3)

Step A: 2-HO-Ac-Ser(Bzl)-Ser(Bzl)-Ser(Bzl)-Chg-Gln-Ser-Leu-PAM Resin(3-1)

Starting with 0.5 mmol (0.67 g) Boc-Leu-PAM resin (Applied BiosystemsInc.-ABI), the protected peptide was synthesized on a 430A ABI peptidesynthesizer. The protocol used a 4 fold excess (2 mmol) of each of thefollowing protected amino acids: Boc-Ser(OBzl), Boc-Gln, Boc-Chg.Coupling was achieved using DCC and HOBT activation inmethyl-2-pyrrolidinone. Removal of the Boc group was performed using 50%TFA in methylene chloride and the TFA salt neutralized withdiisopropylethylamine. 2-Hydroxyacetic acid was used for theintroduction of the N terminal blocking group, which was also carriedout on the peptide synthesizer. At the completion of the synthesis, thepeptide resin was dried to provide the title resin-peptide conjugate.

Step B: 2-HO-Ac-Ser-Ser-Ser-Chg-Ser-Leu-OH (3-2)

The protected peptide resin (3-1), 1.2 g, was treated with HF (15 ml)for 1 hr at 0° C. in the presence of anisole (1.5 ml). After evaporationof the HF, the residue was washed with ether 3 times, and extracted with20% HOAc. The crude peptide products from the HF-cleavage afterlyophilization were purified by preparatory HPLC on a Delta-Pak C18column with 0.1% trifluoroacetic acid -aqueous acetonitrile solventsystems using 100-70% 0.1 %TFA-H₂O, 60 min linear gradient. Fractionscontaining product of at least 99% (HPLC) purity were combined toprovide the title blocked peptide.

FABMS: 804.85 Peptide Content: 1.03 NMOle/mg. HPLC: 99% pure @ 214,retention times = 11.16 min, (Vydac C₁₈, gradient of 95% A/B to 50% A/Bover 30 min, A = 0.1% TFA-H₂O, B = 0.1% TFA-CH₃CN)

Step C: 2-HO-Ac-Ser-Ser-Ser-Chg-Ser-Leu-Dox (3-3)

A solution of 241 mg (0.30 mmol) of OH-Ac-Ser-Ser-Ser-Chg-Gln-Leu-OH(3-2) in 3.0 ml anhyd. N-methyl pyrrolidine (NMP) (or DMF), 46 mg (0.30mmol) of HOBT, 63 mg (0.33 mmol) of EDC, 46 mg (0.09 mmol) ofdoxorubicin was added and pH was adjusted with diisopropylethylamine(DIEA) to pH 8.5. The solution was stirred at 0° C. for 11 hrs., andthen reaction was quenched by H⁺. The organic solvent was removed underreduced pressure and the residue was diluted with 15 ml of water, andpurified by preparative HPLC using a NH₄Ac (4 g/4L)-CH₃CN gradient, ie.95-50%A, 60 min. Lyophilization of pure fractions gave a red powder. Thered powder was dissolved in distil. H₂O, filtered, and lyophilized toprovide the title conjugate (1-3).

ES⁺ + NH₄ ⁺: 1347.61 Peptide Content: 541.72 NMOle/mg. HPLC: 99% pure @214, retention times = 20.8 min, (Vydac C₁₈, gradient of 95% A/B to 50%A/B over 30 min, A = 0.1% TFA-H₂O, B = 0.1% TFA, CH₃CN)

Example 4

Preparation ofN-[2-{2-(2-methoxyethoxy)ethoxy}acetyl]-Ser-Ser-Ser-Chg-Gln-Ser-Leu-Dox

The title conjugate was prepared in the manner described in Example 3,but substituting 2-{2-(2-methoxyethoxy)ethoxy}acetic acid for2-hydroxyacetic acid in Step A.

ES⁺ + NH₄ ⁺: 1450.72 Peptide Content: 534.36 NMOle/mg. HPLC: 99% pure @214, retention times = 21.99 min, (Vydac C₁₈, gradient of 95% A/B to 50%A/B over 30 min, A = 0.1% TFA-H₂O, B = 0.1 %TFA, CH₃CN)

Example 5 Preparation ofN-2(R)-2,3-dihydroxypropionyl-Ser-Ser-Ser-Chg-Gln-Ser-Leu-Dox (5-3) StepA:N-2(R)-2,3-dihydroxypropionyl-Ser(Bzl)-Ser(Bzl)-Ser(Bzl)-Chg-Gln-Ser-Leu-PAMResin (5-1).

Starting with 0.5 mmol (0.67 g) Boc-Leu-PAM resin, the protected peptidewas synthesized on a 430A ABI peptide synthesizer. The protocol used a 4fold excess (2 mmol) of each of the following protected amino acids:Boc-Ser(OBzl), Boc-Gln and Boc-Chg. Coupling was achieved using DCC andHOBT activation in methyl-2-pyrrolidinone. Removal of the Boc group wasperformed using 50% TFA in methylene chloride and the TFA saltneutralized with diisopropylethylamine. D-Glyceric acid, which wasconverted from D-Glyceric acid calcium salt, was used for theintroduction of the N terminal blocking group. At the completion of thesynthesis, the peptide resin was dried to provide the titleresin-peptide conjugate.

Step B: N-2(R)-2,3-dihydroxypropionyl-Ser-Ser-Ser-Chg-Gln-Ser-Leu-Dox(5-3)

The title conjugate was prepared in the manner described in Example 3,Steps B and C, but substituting the resin peptide conjugate 5-1 for theresin-peptide conjugate used in Example 3, Step B.

ES⁺ + NH₄ ⁺: 1377.55 Peptide Content: 620.85 NMOle/mg. HPLC: 99% pure @214, retention times = 20.71 min, (Vydac C₁₈, gradient of 95% A/B to 50%A/B over 30 min, A = 0.1% TFA-H₂O, B = 0.1% TFA, CH₃CN)

Example 6 Preparation ofN-2(S)-2,3-dihydroxypropionyl-Ser-Ser-Ser-Chg-Gln-Ser-Leu-Dox

The title conjugate was prepared in the manner described in Example 5,but substituting L-glyceric acid for D-glyceric acid in Step A.

ES⁺ + NH₄ ⁺: 1377.62 Peptide Content: 641.59 NMOle/mg. HPLC: 99% pure @214, retention times = 20.57 min, (Vydac C₁₈, gradient of 95% A/B to 50%A/B over 30 min, A = 0.1% TFA-H₂O, B = 0.1% TFA, CH₃CN)

Table 3 shows other blocked peptide-doxorubicin conjugates that wereprepared by the procedures described in Examples 3-6, but utilizing theappropriate amino acid residues and blocking group acylation.

TABLE 3 Time to 50% Substrate SEQ. Cleavage by ID. York PSA NO.PEPTIDE/PEPTIDE-DOX CONJUGATE (Min) 642-hydroxyacetyl-HomoRSSYQ-SNle-DOX (3′)  60* 662-hydroxyacetyl-SHomoRChgQ-SL-DOX (3′)  15 672-hydroxyacetyl-HomoRSSChgQ-SL-DOX (3′)  12 682-hydroxyacetyl-HomoRASChgQ-SL-DOX (3′) 10 69(d)2,3-dihydroxypropionyl-SHomoRChgQ-SL-  65 DOX (3′) 70(l)2,3-dihydroxypropionyl-SHomoRChgQ-SL-  15 DOX (3′) 71PEG(2)-SHomoRChgQ-SL-DOX (3′)  25 72 PEG(2)-HomoRChgQ-SL-DOX (3′) 4 HOUR=  12% 73 (2R,3S) 2,3,4-trihydroxybutanoyl-HomoRChgQ-SL- 4 HOUR = DOX(3′)   0% 74 PEG(2)-SHomoRYQ-SL-DOX(3′)  35 75 PEG(2)-HomoRYQ-SSSL-DOX(3′) 4 HOUR =  40% (PS) 76 PEG(2)-KYQ-SSSL-DOX (3′) 4 HOUR =  20% (PS)77 2-hydroxyacetyl-HomoRSSYQ-SL-DOX (3′)  16 (PS) 78(l)2,3-dihydroxypropionyl-HomoRSSChgQ-SL-  12 DOX (3′) 79PEG(2)-HomoRSSChgQ-SL-DOX (3′)  11 80 2-hydroxyacetyl-SYQ-SSSL-DOX (3′)(PS) 81 PEG(16)-SHomoRYQ-SL-DOX (3′)  65 82 (2R,3S)2,3,4-trihydroxybutanoyl-SHomoRChgQ-  45 SL-DOX (3′) 83PEG(2)-SHomoRYQ-SL-DOX (3′)  60 84(d)2,3-dihydroxypropionyl-HomoRSSChgQSL-  12 DOX (3′) 85(l)2,3-dihydroxypropionylSSSChgQ-S(dL)- 180 DOX (3′) 86(d)2,3-dihydroxypropionylSSSChgQ-SL-  55 DOX (3′) 87(l)2,3-dihydroxypropionylSSSChgQ-SL-  25 DOX (3′) 88(l)2,3-dihydroxypropionylSSChgQ-S(dL)- 3 HOUR = DOX (3′)  22% 89(d)2,3-dihydroxypropionylSSChgQ-SL-DOX (3′) 120 91 PEG(2)SSChgQ-SL-DOX(3′)  90 92 PEG(2)-SSSChgQ-S(dL)-DOX (3′) 3 HOURS =  46% 63PEG(2)-SSSChgQ-SL-DOX (3′)  60 94(d)2,3-dihydroxypropionyl-3PALSSChgQ-SL-  12 (PS) DOX (3′).AcOH 95(l)2,3-dihydroxypropionyl-SSChgQ-SL-DOX (3′)  25 612-hydroxyacetyl-SSSChgQ-SL-DOX (3′)  25 962,3-dihydroxypropionyl-HomoSSSChgQ-SL-  35 DOX (3′) 97PEG(2)-ASChgQ-SL-DOX (3′)  45 98 PEG(6)-ASChgQ-SL-DOX (3′) 160 622-hydroxyacetyl-SSChgQ-SL-DOX (3′)  45

Example 7

Assessment of the Recognition of Oligopeptide-Doxorubicin Conjugates byFree PSA

The conjugates prepared as described in Examples 3-6 were individuallydissolved in PSA digestion buffer (50 mMtris(hydroxymethyl)-aminomethane pH7.4, 140 mM NaCl) and the solutionadded to PSA at a molar ration of 100 to 1. The reaction is quenchedafter various reaction times by the addition of trifluoroacetic acid(TFA) to a final 1% (volume/volume). The quenched reaction was analyzedby HPLC on a reversed-phase C18 column using an aqueous 0.1%TFA/acetonitrile gradient. The results of the assessment are shown inTable 3. Table 3 shows the amount of time (in minutes) required for 50%cleavage of the noted oligopeptide-cytotoxic agent conjugates withenzymatically active free PSA. If no salt is indicated for theconjugate, the free conjugate was tested. An alternative PSA digestionbuffer (12 mM tris(hydroxymethyl)-aminomethane pH8.0, 25 mM NaCl, 0.5 mMCaCl₂) was utilized in the assessment of the2-hydroxyacetyl-hArgSerSerTyrGln-SerNle-DOX (3′) (SEQ.ID.NO.: 30)conjugate.

Example 8

In vitro Assay of Cytotoxicity of Peptidyl Derivatives of Doxorubicin

The cytotoxicities of the cleaveable oligopeptide-doxorubicinconjugates, prepared as described in Examples 3-6, against a line ofcells which is known to be killed by unmodified doxorubicin was assessedwith an Alamar Blue assay. Specifically, cell cultures of LNCap prostatetumor cells or DuPRO cells in 96 well plates was diluted with mediumcontaining various concentrations of a given conjugate (final plate wellvolume of 200 μl ). The cells were incubated for 3 days at 37° C., 20 μlof Alamar Blue is added to the assay well. The cells were furtherincubated and the assay plates were read on a EL-310 ELISA reader at thedual wavelengths of 570 and 600 nm at 4 and 7 hours after addition ofAlamar Blue. Relative percentage viability at the various concentrationof conjugate tested was then calculated versus control (no conjugate)cultures. Results of this assay are shown in Table 4. If no salt isindicated, the free conjugate was tested.

TABLE 4 SEQ. LNCaP Cell Kill in ID. PEPTIDE/PEPTIDE- 72 HRS, {48 HRS}NO. DOX CONJUGATE EC 50 (μM) 64 2-hydroxyacetyl-hRSSYQ-SNle- 3.6(DuPRO > 100) DOX (3′) 66 2-hydroxyacetyl-ShRChgQ-SL- 5.1 (DUPRO > 100)DOX (3′) 67 2-hydroxyacetyl-hRSSChgQ-SL- 5.5 (DuPRO > 100) DOX (3′) 682-hydroxyacetyl-hRASChgQ-SL- 7.9 (DuPRO > 100) (PS) DOX (3′) 69(d)2,3-dihydroxypropionyl- 5.8 (DuPRO > 100) n = 2 ShRChgQ-SL-DOX (3′)70 (l)2,3-dihydroxypropionyl- 9.4 (DuPRO > 100) n = 2 ShRChgQ-SL-DOX(3′) 71 PEG(2)-ShRChgQ-SL-DOX (3′) 8.1 (DuPRO > 100) 72PEG(2)-hRChgQ-SL-DOX (3′) INSOLUBLE 73 (2R,3S) 2,3,4-trihydroxybutanoyl-PS hRChgQ-SL-DOX (3′) 74 PEG(2)-ShRYQ-SL-DOX(3′) 4.5 (DuPRO > 100) 75PEG(2)-hRYQ-SSSL-DOX (3′) 14 (DuPRO > 100) (PS) 76 PEG(2)-KYQ-SSSL-DOX(3′) 12.8 (DuPRO > 100) (PS) 77 2-hydroxyacetyl-hRSSYQ-SL- 13.6 (DuPRO >100) (PS) DOX (3′) 78 (l)2,3-dihydroxypropionyl- 7.5 (DuPRO > 100)hRSSChgQSL-DOX (3′) 79 PEG(2)-hRSSChgQ-SL-DOX (3′) 5.7 (DuPRO > 100) 802-hydroxyacetyl-SYQ-SSSL- 18.8 (DuPRO = 50) (PS) DOX (3′) 81PEG(16)-ShRYQ-SL-DOX (3′) 45 (DuPRO = 100) 82 (2R,3S)2,3,4-trihydroxybutanoyl- 14.1 (DuPRO > 100) ShRChgQ-SL-DOX (3′) 83PEG(2)-ShRYQ-SL-DOX (3′) 34 (DuPRO = 100) n = 2 84(d)2,3-dihydroxypropionyl- 7.7 (DuPRO > 10o) n = 2 hRSSChgQSL-DOX(3′) 85(l)2,3-dihydroxypropionyl- 91 (DuPRO > 100) SSSChgQ-S(dL)-DOX (3′) 86(d)2,3-dihydroxypropionyl- 5.8 (DuPRO > 100) n = 3 SSSChgQ-SL-DOX (3′)87 (l)2,3-dihydroxypropionyl- 5.5 (DuPRO > 100) SSSChgQ-SL-DOX (3′) 88(l)2,3-dihydroxypropionyl- >100 (DuPRO > 100) SSChgQ-S(dL)-DOX (3′) 89(d)2,3-dihydroxypropionyl- 9.1 (DuPRO > 100) SSChgQ-SL-DOX (3′) 91PEG(2)SSChgQ-SL-DOX (3′) 8.8 (DuPRO > 100) 63 PEG(2)-SSSChgQ-SL-DOX (3′)10 (DuPRO > 100) n = 2 94 (d)2,3-dihydroxypropionyl-3PAL- 5.5 (DuPRO >100) SSChgQ-SL-DOX (3′).AcOH 95 (l)2,3-dihydroxypropionyl- 13 (DuPRO >100) n = 2 SSChgQ-SL-DOX (3′) 61 2-hydroxyacetyl-SSSChgQ- 7.2 (DuPRO >100) n = 3 SL-DOX (3′) 96 2,3-dihydroxypropionyl- 5.1 (DuPRO = 90)hSSSChgQ-SL-DOX (3′) 97 PEG(2)-ASChgQ-SL-DOX (3′) 5.6 (DuPRO = 100) n =2 98 PEG(6)-ASChgQ-SL-DOX (3′) 12 (DuPRO = 100) 622-hydroxyacetyl-SSChgQ- 4.8 (DuPRO > 100) SL-DOX (3′)

Example 9

In vivo Efficacy of Peptidyl-Cytotoxic Agent Conjugates

LNCaP.FGC or DuPRO-1 cells are trypsinized, resuspended in the growthmedium and centifuged for 6 mins. at 200×g. The cells are resuspended inserum-free α-MEM and counted. The appropriate volume of this solutioncontaining the desired number of cells is then transferred to a conicalcentrifuge tube, centrifuged as before and resuspended in theappropriate volume of a cold 1:1 mixture of α-MEM-Matrigel. Thesuspension is kept on ice until the animals are inoculated.

Harlan Sprague Dawley male nude mice (10-12 weeks old) are restrainedwithout anesthesia and are inoculated with 0.5 mL of cell suspension onthe left flank by subcutaneous injection using a 22 G needle. Mice areeither given approximately 5×10⁵ DuPRO cells or 1.5×10⁷ LNCaP.FGC cells.

Following inoculation with the tumor cells the mice are treated underone of two protocols:

Protocol A

One day after cell inoculation the animals are dosed with a 0.1-0.5 mLvolume of test conjugate, doxorubicin or vehicle control (sterilewater). Dosages of the conjugate and doxorubicin are initially themaximum non-lethal amount, but may be subsequently titrated lower.Identical doses are administered at 24 hour intervals for 5 days. After10 days, blood samples are removed from the mice and the serum level ofPSA is determined. Similar serum PSA levels are determined at 5-10 dayintervals. At the end of 5.5 weeks the mice are sacrificed and weightsof any tumors present are measured and serum PSA again determined. Theanimals' weights are determined at the beginning and end of the assay.

Protocol B

Ten days after cell inoculation, blood samples are removed from theanimals and serum levels of PSA are determined. Animals are then groupedaccording to their PSA serum levels. At 14-15 days after cellinoculation, the animals are dosed with a 0.1-0.5 mL volume of testconjugate, doxorubicin or vehicle control (sterile water). Dosages ofthe conjugate and doxorubicin are initially the maximum non-lethalamount, but may be subsequently titrated lower. Identical doses areadministered at 24 hour intervals for 5 days. Serum PSA levels aredetermined at 5-10 day intervals. At the end of 5.5 weeks the mice aresacrificed, weights of any tumors present are measured and serum PSAagain determined. The animals' weights are determined at the beginningand end of the assay.

Example 10

In vitro Determination of Proteolytic Cleavage of Conjugates byEndogenous Non-PSA Proteases

Step A: Preparation of Proteolytic Tissue Extracts

All procedures are carried out at 4° C. Appropriate animals aresacrificed and the relevant tissues are isolated and stored in liquidnitrogen. The frozen tissue is pulverized using a mortar and pestle andthe pulverized tissue is transfered to a Potter-Elvejeh homogenizer and2 volumes of Buffer A (50 mM Tris containing 1.15% KCl, pH 7.5) areadded. The tissue is then disrupted with 20 strokes using first a losefitting and then a tight fitting pestle. The homogenate is centrifugedat 10,000×g in a swinging bucket rotor (HB4-5), the pellet is discardedand the re-supernatant centrifuged at 100,000×g (Ti 70). The supernatant(cytosol) is saved.

The-pellet is respuspended in Buffer B (10 mM EDTA containing 1.15% KCl,pH 7.5) using the same volume used in step as used above with Buffer A.The suspension is homogenized in a dounce homogenizer and the solutioncentrifuged at 100,000×g. The supernatant is discarded and the pelletresuspended in Buffer C (10 mM potassium phosphate buffer containing0.25 M sucrose, pH 7.4), using ½ the volume used above, and homogenizedwith a dounce homogenizer.

Protein content of the two solutions (cytosol and membrane) is determineusing the Bradford assay. Assay aliquots are then removed and frozen inliquid N₂. The aliquots are stored at −70° C.

Step B: Proteolytic Cleavage Assay

For each time point, 20 microgram of peptide-doxorubicin conjugate and150 micrograms of tissue protein, prepared as described in Step A and asdetermined by Bradford in reaction buffer are placed in solution offinal volume of 200 microliters in buffer (50 mM TRIS, 140 mM NaCl, pH7.2). Assay reactions are run for 0, 30, 60, 120, and 180 minutes andare then quenched with 9 microliters of 0.1 M ZnCl₂ and immediatelyplaced in boiling water for 90 seconds. Reaction products are analyzedby HPLC using a VYDAC C18 15 cm column in water/acetonitrile (5% to 50%acetonitrile over 30 minutes).

What is claimed is:
 1. A conjugate which is useful for the treatment ofprostate cancer which comprises a cytotoxic agent attached to aoligopeptide, wherein the oligopeptide comprises a sequence of aminoacids that is selectively proteolytically cleaved by free prostatespecific antigen, wherein the means of attachment is a covalent bond orthrough a chemical linker and wherein the point of attachment on theoligopeptide is at the C-terminus, and which further comprises ahydrophilic blocking group at the N-terminus of the oligopeptide, or thepharmaceutically acceptable salt thereof.
 2. The conjugate according toclaim 1 wherein the cytotoxic agent is a member of a class of cytotoxicagents selected from the following classes: a) anthracycline family ofdrugs, b) the vinca alkaloid drugs, c) the mitomycins, d) thebleomycins, e) the cytotoxic nucleosides, f) the pteridine family ofdrugs, g) diynenes, h) estramustine, i) cyclophosphamide, j) the taxanesand k) the podophyllotoxins, or the pharmaceutically acceptable saltthereof.
 3. The conjugate according to claim 2 wherein the cytotoxicagent is selected from the following cytotoxic agents: a) doxorubicin,b) carminomycin, c) daunorubicin, d) aminopterin, e) methotrexate, f)methopterin, g) dichloro-methotrexate, h) mitomycin C, i) porfiromycin,j) 5-fluorouracil, k) 6-mercaptopurine, l) cytosine arabinoside, m)podophyllotoxin, n) etoposide, o) etoposide phosphate, p) melphalan, q)vinblastine, r) vincristine, s) leurosidine, t) vindesine, u)estramustine, v) cisplatin, w) cyclophosphamide, x) taxol, and y)leurosine, or the pharmaceutically acceptable salt thereof.
 4. Theconjugate according to claim 2 wherein the cytotoxic agent is selectedfrom doxorubicin and vinblastine or a cytotoxic derivative thereof. 5.The conjugate according to claim 2 wherein the cytotoxic agent isdoxorubicin or a cytotoxic derivative thereof.
 6. The conjugateaccording to claim 1 wherein the oligopeptide comprises an oligomerselected from: a) AsnLysIleSerTyrGln|Ser (SEQ.ID.NO.: 1), b)LysIleSerTyrGln|Ser (SEQ.ID.NO.: 2), c) AsnLysIleSerTyrTyr|Ser(SEQ.ID.NO.: 3), d) AsnLysAlaSerTyrGln|Ser (SEQ.ID.NO.: 4), e)SerTyrGln|SerSer (SEQ.ID.NO.: 5); f) LysTyrGln|SerSer (SEQ.ID.NO.: 6);g) hArgTyrGln|SerSer (SEQ.ID.NO.: 7); h) hArgChaGln|SerSer (SEQ.ID.NO.:8); i) TyrGln|SerSer (SEQ.ID.NO.: 9); j) TyrGln|SerLeu (SEQ.ID.NO.: 10);k) TyrGln|SerNle SEQ.ID.NO.: 11); l) ChgGln|SerLeu (SEQ.ID.NO.: 12); andm) ChgGln|SerNle (SEQ.ID.NO.: 13).
 7. The conjugate according to claim 1wherein the oligopeptide comprises an oligomer selected from: a)AsnLysIleSerTyrGln|SerSer (SEQ.ID.NO.: 14), b) AsnLysIleSerTyrGln|SerAla(SEQ.ID.NO.: 15), c) AlaAsnLysIleSerTyrTyr|Ser (SEQ.ID.NO.: 16), d)AlaAsnLysAlaSerTyrGln|Ser (SEQ.ID.NO.: 17), e) SerTyrGln|SerSerThr(SEQ.ID.NO.: 18), f) SerTyrGln|SerSerSer (SEQ.ID.NO.: 19), g)LysTyrGln|SerSerSer (SEQ.ID.NO.: 20), h) hArgTyrGln|SerSerSer(SEQ.ID.NO.: 21), i) SerTyrGln|SerSerLeu (SEQ.ID.NO.: 22); j)SerTyrGln|SerLeu (SEQ.ID.NO.: 23); k) SerChgGln|SerLeu (SEQ.ID.NO.: 24);l) hArgChgGln|SerLeu (SEQ.ID.NO.: 25); and m) hArgTyrGln|SerLeu(SEQ.ID.NO.: 26).
 8. The conjugate according to claim 1 wherein theoligopeptide comprises an oligomer selected from:GlyGluAsnGlyValGlnLysAspValSerGlnArgSerIleTyr|SerGlnThrGlu (SEQ.ID.NO.:27), AlaSerTyrGln|SerSerLeu (SEQ.ID.NO.: 28); SerhArgChgGln|SerLeu(SEQ.ID.NO.: 29); hArgSerSerTyrGln|SerNle (SEQ.ID.NO.: 30);hArgAlaSerChgGln|SerLeu (SEQ.ID.NO.: 31); hArgSerSerTyrGln|SerLeu(SEQ.ID.NO.: 32); hArgSerSerChg|SerLeu (SEQ.ID.NO.: 33);SerhArgChgGln|SerLeu (SEQ.ID.NO.: 34); hArgTyrGln|SerLeu (SEQ.ID.NO.:35); hArgSerSerChgGln|SerLeu (SEQ.ID.NO.: 36); SerhArgTyrGln|SerLeu(SEQ.ID.NO.: 37); SerSerTyrGln|SerLeu (SEQ.ID.NO.: 38);SerSerSerChgGln|SerLeu (SEQ.ID.NO.: 39); 3PAL-SerSerChgGln|SerLeu(SEQ.ID.NO.: 40); SerSerChgGln|SerLeu (SEQ.ID.NO.: 41);SerSerSerChgGln|Ser(dLeu) (SEQ.ID.NO.: 42); SerSerSerChgGln|SerVal(SEQ.ID.NO.: 43); ProSerSerChgGln|SerVal (SEQ.ID.NO.: 44);GlySerSerChgGln|SerLeu (SEQ.ID.NO.: 45); hSerSerSerChgGln|SerLeu(SEQ.ID.NO.: 46); hArgSerSerChgGln|SerNle (SEQ.ID.NO.: 47);hArgTyrGln|SerSerSerLeu (SEQ.ID.NO.: 55); LysTyrGln|SerSerSerLeu(SEQ.ID.NO.: 56); SerTyrGln|SerSerSerLeu (SEQ.ID.NO.: 57);SerSerChgGln-Ser(dLeu) (SEQ.ID.NO.: 58); and 3PAL-SerSerChgGln-Ser(dLeu)(SEQ.ID.NO.: 59); and AlaSerChgGln-SerLeu (SEQ.ID.NO.: 60).
 9. Theconjugate according to claim 1 wherein the hydrophilic blocking group isselected from:

wherein: R¹ and R² are independently selected from: a) hydrogen, b)unsubstituted or substituted aryl, unsubstituted or substitutedheterocycle, C₃-C₁₀ cycloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, halogen,C₁-C₆ perfluoroalkyl, R¹²O—, R³C(O)NR³—, (R³)₂NC(O)—, R³ ₂N—C(NR³)—,R⁴S(O)_(m)NH, CN, NO₂, R³C(O)—, N₃, —N(R³)₂, or R⁴OC(O)NR³—, c)unsubstituted C₁-C₆ alkyl, d) substituted C₁-C₆ alkyl wherein thesubstituent on the substituted C₁-C₆ alkyl is selected fromunsubstituted or substituted aryl, unsubstituted or substitutedheterocyclic, C₃-C₁₀ cycloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, R³O—,R⁴S(O)_(m)NH, R³C(O)NR³—, (R³)₂NC(O)—, R³ ₂N—C(NR³)—, CN, R³C(O)—, N₃,—N(R³)₂, and R⁴OC(O)—NR³—; or R¹ and R² are combined to form —(CH₂)_(s)—wherein one of the carbon atoms is optionally replaced by a moietyselected from: O, S(O)_(m), —NC(O)—, NH and —N(COR¹⁰)—; R³ is selectedfrom: hydrogen, aryl, substituted aryl, heterocycle, substitutedheterocycle, C₁-C₆ alkyl and C₃-C₁₀ cycloalkyl; R⁴ is selected from:aryl, substituted aryl, heterocycle, substituted heterocycle, C₁-C₆alkyl and C₃-C₁₀ cycloalkyl; m is 0, 1 or 2; n is 1, 2, 3 or 4; p iszero or an integer between 1 and 100; and q is 0 or 1, provided that ifp is zero, q is 1; and s is 3, 4 or
 5. 10. A conjugate which is usefulfor the treatment of prostate cancer of the formula I:

wherein: oligopeptide is an oligopeptide which is selectively recognizedby the free prostate specific antigen (PSA) and is capable of beingproteolytically cleaved by the enzymatic activity of the free prostatespecific antigen, and wherein the C-terminus carbonyl is covalentlybound to the amine of doxorubicin and the N-terminus amine is covalentlybound to the carbonyl of the blocking group; R is selected from

R¹ and R² are independently selected from: hydrogen, OH, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ aralkyl and aryl; n is 1, 2, 3 or 4; p is zero or aninteger between 1 and 100; q is 0 or 1, provided that if p is zero, q is1; or the pharmaceutically acceptable salt thereof.
 11. The conjugateaccording to claim 10 wherein: R is selected from

R¹ and R² are independently selected from: hydrogen, C₁-C₆ alkyl andaryl; n is 1, 2, 3 or 4; n′ is 0, 1, 2 or 3; p is zero or an integerbetween 1 and 14; q is 0 or 1, provided that if p is zero, q is 1; orthe pharmaceutically acceptable salt thereof.
 12. The conjugateaccording to claim 10 wherein: oligopeptide is an oligomer thatcomprises an amino acid sequence selected from: a)AsnLysIleSerTyrGln|Ser (SEQ.ID.NO.: 1), b) LysIleSerTyrGln|Ser(SEQ.ID.NO.: 2), c) AsnLysIleSerTyrTyr|Ser (SEQ.ID.NO.: 3), d)AsnLysAlaSerTyrGln|Ser (SEQ.ID.NO.: 4), e) SerTyrGln|SerSer (SEQ.ID.NO.:5); f) LysTyrGln|SerSer (SEQ.ID.NO.: 6); g) hArgTyrGln|SerSer(SEQ.ID.NO.: 7); h) hArgChaGln|SerSer (SEQ.ID.NO.: 8); i) TyrGln|SerSer(SEQ.ID.NO.: 9); j) TyrGln|SerLeu (SEQ.ID.NO.: 10); k) TyrGln|SerNle(SEQ.ID.NO.: 11); l) ChgGln|SerLeu (SEQ.ID.NO.: 12); m) ChgGln|SerNle(SEQ.ID.NO.: 13); or an optical isomer or pharmaceutically acceptablesalt thereof.
 13. The conjugate according to claim 10 wherein:oligopeptide is an oligomer that comprises an amino acid sequenceselected from:GlyGluAsnGlyValGlnLysAspValSerGlnArgSerIleTyr|SerGlnThrGlu (SEQ.ID.NO.:27), AlaSerTyrGLn|SerSerLeu (SEQ.ID.NO.: 28); SerhArgChgGln|SerLeu(SEQ.ID.NO.: 29); hArgSerSerTyrGln|SerNle (SEQ.ID.NO.: 30);hArgAlaSerChgGln|SerLeu (SEQ.ID.NO.: 31); hArgSerSerTyrGln|SerLeu(SEQ.ID.NO.: 32); hArgSerSerChg|SerLeu (SEQ.ID.NO.: 33);SerhArgChgGln|SerLeu (SEQ.ID.NO.: 34); hArgTyrGln|SerLeu (SEQ.ID.NO.:35); hArgSerSerChgGln|SerLeu (SEQ.ID.NO.: 36); SerhArgTyrGln|SerLeu(SEQ.ID.NO.: 37); SerSerTyrGln|SerLeu (SEQ.ID.NO.: 38);SerSerSerChgGln|SerLeu (SEQ.ID.NO.: 39); 3PAL-SerSerChgGln|SerLeu(SEQ.ID.NO.: 40); SerSerChgGln|SerLeu (SEQ.ID.NO.: 41);SerSerSerChgGln|Ser(dLeu) (SEQ.ID.NO.: 42); SerSerSerChgGln|SerVal(SEQ.ID.NO.: 43); ProSerSerChgGln|SerVal (SEQ.ID.NO.: 44);GlySerSerChgGln|SerLeu (SEQ.ID.NO.: 45); hSerSerSerChgGln|SerLeu(SEQ.ID.NO.: 46); hArgSerSerChgGln|SerNle (SEQ.ID.NO.: 47);hArgTyrGln|SerSerSerLeu (SEQ.ID.NO.: 55); LysTyrGln|SerSerSerLeu(SEQ.ID.NO.: 56); SerTyrGln|SerSerSerLeu (SEQ.ID.NO.: 57);SerSerChgGln-Ser(dLeu) (SEQ.ID.NO.: 58); and 3PAL-SerSerChgGln-Ser(dLeu)(SEQ.ID.NO.: 59); and AlaSerChgGln-SerLeu (SEQ.ID.NO.: 60) or an opticalisomer or pharmaceutically acceptable salt thereof.
 14. The conjugateaccording to claim 1 of the formula II:

wherein: oligopeptide is an oligopeptide which is specificallyrecognized by the free prostate specific antigen (PSA) and is capable ofbeing proteolytically cleaved by the enzymatic activity of the freeprostate specific antigen and wherein the point of attachment of theoligopeptide to X_(L) is at the C-terminus; X_(L) is —NH—(CH₂)_(r)—NH— Ris selected from

 R₁ and R₂ are independently selected from: hydrogen, OH, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ aralkyl and aryl; n is 1, 2, 3 or 4; p is zero or aninteger between 1 and 100; q is 0 or 1, provided that if p is zero, q is1; r is 1, 2, 3, 4 or 5, or a pharmaceutically acceptable salt thereof.15. A conjugate of the formula III:

wherein: oligopeptide is an oligopeptide which is specificallyrecognized by the free prostate specific antigen (PSA) and is capable ofbeing proteolytically cleaved by the enzymatic activity of the freeprostate specific antigen, R^(d) and R^(e) are independently selectedfrom: hydrogen, C₁-C₆-alkyl, —C₁-C₆-alkyl-OH, —C₁-C₆-alkyl-di-OH,—C₁-C₆-alkyl-tri-OH and

 provided that at least one R^(d) and R^(e) are not hydrogen orC₁-C₆-alkyl, or R^(d) and R^(e) are combined to form a —CH₂CH₂OCH₂CH₂—diradical; p is zero or an integer between 1 and 100; q is 0 or 1,provided that if p is zero, q is 1; or a pharmaceutically acceptablesalt thereof.
 16. A pharmaceutical composition comprising apharmaceutical carrier, and dispersed therein, a therapeuticallyeffective amount of a compound of claim
 1. 17. A pharmaceuticalcomposition comprising a pharmaceutical carrier, and dispersed therein,a therapeutically effective amount of a compound of claim
 10. 18. Amethod for treating prostate cancer which comprises administering to amammal in need thereof a therapeutically effective amount of acomposition of claim
 16. 19. A method for treating prostate cancer whichcomprises administering to a mammal in need thereof a therapeuticallyeffective amount of a composition of claim
 17. 20. A method for treatingbenign prostatic hyperplasia which comprises administering to a mammalin need thereof a therapeutically effective amount of a composition ofclaim
 16. 21. A method for treating benign prostatic hyperplasia whichcomprises administering to a mammal in need thereof a therapeuticallyeffective amount of a composition of claim
 17. 22. A pharmaceuticalcomposition made by combining the compound of claim 1 and apharmaceutically acceptable carrier.
 23. A process for making apharmaceutical composition comprising combining a compound of claim 1and a pharmaceutically acceptable carrier.